According to the NIAID fact sheet on Botulism, the extreme toxicity of botulinum neurotoxins (BoNT) and the ease of production, transport, and delivery make this an agent of extreme bioterrorism concern. Nonetheless, although there are major vaccine development initiatives ongoing, there currently is no approved Botulinum toxin vaccine available. Advances in Botulinum research have designated the optimal target for vaccine development to be the non-toxic carboxyterminal half of the toxin heavy chain (HC50). In fact, an immediate research goal for NIAID is listed as the development of a HC50 fragment vaccine against botulinum. Building upon the encouraging data from the phase I proof of concept study of this project, the overall phase II project goal is to develop a new, trivalent botulinum vaccine against the most common serotypes A, B and E. Three Aims are proposed for the further development of this novel trivalent BoNT vaccine as follows: Specific Aim 1: Vaccine vector construction and recovery. i) plasmid constructions, ii) demonstration of cell surface expression of the BoNT HC50, and iii) re-recovery of recombinant RVs from cDNA on Vero. Specific Aim 2. Vaccine vector characterization i) purification, inactivation, and in vitro characterization of the RV virions, ii) analysis of mouse immune responses to the vaccine using ELISA, in vitro neutralization assays, and in vivo protection of mice against a single challenge with BoNT or multiple BoNT challenge dependent on the used vaccine(s) for immunization. Specific Aim 3: Development of Pilot Production and Purification Processes. The overall goal of this aim is to translate the production process of our vaccine from a research setting to one suitable for pilot scale vaccine manufacture.